Amino-terminal amino acid sequences (42 residues) were determined for the products of the three common alleles at the human placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] gene locus. The sequences differ at position 3, which is proline in types 1 and 2 but is leucine in type 3. cDNA libraries were constructed in phage Xgtl1 and used to isolate clones covering the coding regions of types 1 and 3 cDNAs. Comparison of the deduced amino acid sequences of the types 1 and 3 proteins showed 7 differences out of 513 amino acids, each due to a single base substitution. cDNA sequence comparisons showed three silent substitutions in the coding regions and three base differences in the greater than 1 kilobase pairs of 3' untranslated sequences.

Wolfram syndrome is a rare multisystem disease characterized by childhood-onset diabetes mellitus and progressive neurodegeneration. Most cases are attributed to pathogenic variants in a single gene, Wolfram syndrome 1 (WFS1). There currently is no disease-modifying treatment for Wolfram syndrome, as the molecular consequences of the loss of WFS1 remain elusive. Because diabetes mellitus is the first diagnosed symptom of Wolfram syndrome, we aimed to further examine the functions of WFS1 in pancreatic β cells in the context of hyperglycemia. Knockout (KO) of WFS1 in rat insulinoma (INS1) cells impaired calcium homeostasis and protein kinase B/Akt signaling and, subsequently, decreased cell viability and glucose-stimulated insulin secretion. Targeting calcium homeostasis with reexpression of WFS1, overexpression of WFS1’s interacting partner neuronal calcium sensor-1 (NCS1), or treatment with calpain inhibitor and ibudilast reversed deficits observed in WFS1-KO cells. Collectively, our findings provide insight into the disease mechanism of Wolfram syndrome and highlight new targets and drug candidates to facilitate the development of a treatment for this disorder and similar diseases.

No abstract

Using immunoblots, we identified proteins of Borrelia burgdorferi recognized by sera from 62 patients with either acute or chronic Lyme disease. In all groups studied, the 41-kDa flagellar protein and a relatively minor 93-kDa protein (p93) were the most commonly recognized antigens in patients with acute and chronic disease due to B. burgdorferi. A murine monoclonal antibody (MAb 181.1) was developed against p93, and the antigen was detected by immunoblot analysis in four European and American strains of B. burgdorferi. On two-dimensional gel electrophoresis, p93 had an apparent pI of 6.8. Immunoelectronmicroscopy with MAb 181.1 demonstrated that p93 is located within the protoplasmic cylinder compartment of the organism. The gene encoding p93 was retrieved from a phage expression library. The derived amino acid sequence of p93 confirmed chemical characterization of the antigen, including its amino-terminal peptide sequence. The derived amino acid sequence predicted it to be predominantly alpha helical. A prominent antigenic domain located at the carboxy portion of the protein was recognized by human and rabbit polyclonal antisera and human (MAb D4) and mouse (MAb 181.1) MAbs.

Background After chemotherapy, many cancer survivors suffer from long-lasting cognitive impairment, colloquially known as “chemobrain.” However, the trajectories of cognitive changes and the underlying mechanisms remain unclear. We previously established paclitaxel-induced inositol trisphosphate receptor (InsP3R)-dependent calcium oscillations as a mechanism for peripheral neuropathy, which was prevented by lithium pretreatment. Here, we investigated if a similar mechanism also underlay paclitaxel-induced chemobrain. Method Mice were injected with 4 doses of 20 mg/kg paclitaxel every other day to induced cognitive impairment. Memory acquisition was assessed with the displaced object recognition test. The morphology of neurons in the prefrontal cortex and the hippocampus was analyzed using Golgi-Cox staining, followed by Sholl analyses. Changes in protein expression were measured by Western blot. Results Mice receiving paclitaxel showed impaired short-term spatial memory acquisition both acutely 5 days post injection and chronically 23 days post injection. Dendritic length and complexity were reduced in the hippocampus and the prefrontal cortex after paclitaxel injection. Concurrently, the expression of protein kinase C α (PKCα), an effector in the InsP3R pathway, was increased. Treatment with lithium before or shortly after paclitaxel injection rescued the behavioral, cellular, and molecular deficits observed. Similarly, memory and morphological deficits could be rescued by pretreatment with chelerythrine, a PKC inhibitor. Conclusion We establish the InsP3R calcium pathway and impaired neuronal morphology as mechanisms for paclitaxel-induced cognitive impairment. Our findings suggest lithium and PKC inhibitors as candidate agents for preventing chemotherapy-induced cognitive impairment.

Changes in intracellular calcium (Ca 2+ ) signaling can modulate cellular machinery required for cancer progression. Neuronal calcium sensor 1 (NCS1) is a ubiquitously expressed Ca 2+ -binding protein that promotes tumor aggressiveness by enhancing cell survival and metastasis. However, the underlying mechanism by which NCS1 contributes to increased tumor aggressiveness has yet to be identified. In this study, we aimed to determine (a) whether NCS1 expression changes in response to external stimuli, (b) the importance of NCS1 for cell survival and migration, and (c) the cellular mechanism(s) through which NSC1 modulates these outcomes. We found that NCS1 abundance increases under conditions of stress, most prominently after stimulation with the pro-inflammatory cytokine tumor necrosis factor a, in a manner dependent on nuclear factor kappa-light-chain-enhancer of activated B cells (NFjB). We found that NFjB signaling is activated in human breast cancer tissue, which was accompanied by an increase in NCS1 mRNA expression. Further exploration into the relevance of NCS1 in breast cancer progression showed that knockout of NCS1 (NCS1 KO) caused decreased cell survival and motility, increased baseline intracellular Ca 2+ levels, and decreased inositol 1,4,5-trisphosphate-mediated Ca 2+ responses. Protein kinase B (Akt) activity was decreased in NCS1 KO cells, which could be rescued by buffering intracellular Ca 2+ . Conversely, Akt activity was increased in cells overexpressing NCS1 (NCS1 OE). We therefore conclude that NCS1 acts as cellular stress response protein up-regulated by stress-induced NFjB signaling and that NCS1 influences cell survival and motility through effects on Ca 2+ signaling and Akt pathway activation. AbbreviationsAkt, protein kinase B; Ca 2+ , calcium; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; InsP3R, inositol 1,4,5-trisphosphate receptor; IjBa, nuclear factor of kappa-light polypeptide gene enhancer in B-cell inhibition, alpha; NCS1, neuronal calcium sensor 1; NFjB, nuclear factor kappa-light-chain-enhancer of activated B cells; p-Akt, phosphorylated Akt; PI3K, phosphoinositide-3-kinase; PP2Ac, catalytic subunit of protein phosphatase 2A; tBHP, tert-butylhydroperoxide; TG, thapsigargin; TNF-a, tumor necrosis factor alpha.

Polycystin 2 (PC2 or TRPP1, formerly TRPP2) is a calcium-permeant Transient Receptor Potential (TRP) cation channel expressed primarily on the endoplasmic reticulum (ER) membrane and primary cilia of all cell and tissue types. Despite its ubiquitous expression throughout the body, studies of PC2 have focused primarily on its role in the kidney, as mutations in PC2 lead to the development of autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition for which there is no cure. However, the endogenous role that PC2 plays in the regulation of general cellular homeostasis remains unclear. In this study, we measure how PC2 expression changes in different pathological states, determine that its abundance is increased under conditions of cellular stress in multiple tissues including human disease, and conclude that PC2-deficient cells have increased susceptibility to cell death induced by stress. Our results offer new insight into the normal function of PC2 as a ubiquitous stress-sensitive protein whose expression is up-regulated in response to cell stress to protect against pathological cell death in multiple diseases. Polycystin-2 (PC2 or TRPP1, formerly TRPP2) is a Transient Receptor Potential (TRP) channel most well-known for its associated pathology. When mutated, PC2 causes autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition leading to bilateral renal cyst formation and eventual kidney failure 1. Located primarily on the endoplasmic reticulum (ER) and primary cilia of all cell and tissue types 2-5 , PC2 is a calcium (Ca 2+)-permeant cation channel whose expression level directly affects Ca 2+ release from the ER 5. As such, PC2 is thought to play a key role in regulating Ca 2+-regulated homeostasis and signaling pathways 6. This is supported by findings showing that polycystin-deficient cells exhibit dysregulated Ca 2+ mobilization and Ca 2+-regulated signaling pathways 5,7 , including pathologically increased cAMP levels 8,9 and changes in mitochondrial Ca 2+ uptake 10,11. The aberrant Ca 2+ signaling caused by loss of polycystins is therefore often pointed to as a central cause of enhanced apoptosis 12,13 , excess fluid secretion 14,15 , and metabolic abnormalities 10,11,16-18 seen in cystic kidney cells. Given the importance of PC2 in ADPKD development, most studies of PC2 have focused on its function in the kidney. However, the ubiquitous expression of PC2 in all cell types suggests that it is important in maintaining Ca 2+ homeostasis in tissues beyond the kidney. The tight regulation of intracellular Ca 2+ is necessary for many physiological functions, including protecting cells against outside stressors. Oxidative and ER stress responses require Ca 2+ influx from both the extracellular

Wolfram syndrome (WS) is a rare, progressive disorder characterized by childhood-onset diabetes mellitus, optic nerve atrophy, hearing loss, diabetes insipidus, and neurodegeneration. Currently, there is no effective treatment for WS, and patients typically die between 30 and 40 years of age. WS is primarily caused by autosomal recessive mutations in the Wolfram syndrome 1 (WFS1) gene (OMIM 222300), which encodes for wolframin (WFS1). This disorder is therefore a valuable monogenic model for prevalent diseases, particularly diabetes mellitus and neurodegeneration. Whereas reduced survival and secretion are known cellular impairments causing WS, the underlying molecular pathways and the physiological function of WFS1 remain incompletely described. Here, we characterize WFS1 as a regulator of intracellular calcium homeostasis, review our current understanding of the disease mechanism of WS, and discuss candidate treatment approaches. These insights will facilitate identification of new therapeutic strategies not only for WS but also for diabetes mellitus and neurodegeneration.